The long-range goals of the proposed research are to identify the components of the anion-selective pore of the cystic fibrosis transmembrane conductance regulator (CFTR), to understand the physical basis for anion conduction and to discover the moving parts of the anion conduction pathway. Anion conduction through the CFTR chloride channel is a key element in two devastating diseases, cystic fibrosis and secretory diarrhea. Modifying CFTR channel activity could significantly impact therapies, but neither the structural basis for anion conduction nor the role of the transmembrane segments in gating has been fully defined. Covalent labeling of engineered cysteines has been used to identify elements of the anion conduction pathway of CFTR, but three factors limit the utility of this approach. First, conventional thiol-directed reagents cannot probe the narrowest portion of the pore. Second, our recent results identify spontaneous and potentially confounding reactions of cysteine thiols not fully appreciated in previous studies. Third, the lack of an atomic- scale model for the pore has prevented any realistic understanding of the physical basis for anion conduction. Our recent work suggests new approaches. We have demonstrated that the permeant, pseudohalide anions, [Au(CN)2]- and [Ag(CN)2]- can be locked inside the CFTR pore by means of a ligand substitution reaction. These permeant probes have provided access to engineered cysteines that are invisible to more conventional reagents. Cysteines substituted at these conformationally restricted sites exhibit dramatic changes in reactivity that correlate with the channel gating state. We have also carried out the first exhaustive survey of the reactivity of cysteines engineered into CFTR and defined spontaneous reactions that dramatically alter thiol reactivity. We have implemented an expanded panel of thiol-reactive reagents that will be used to chemically fingerprint engineered cysteines and control for confounding thiol reactions. Finally, collaboration with Mark Sansom has resulted in the first atomic-scale, homology models for the CFTR pore. The specific aims of the proposed research are: 1. To operationally define different regions of the conduction path by comparing quantitatively the reactivity of substituted cysteines toward a panel of permeant and impermeant, thiol-reactive probes differing in size, charge, and reaction mechanism. 2. To identify a subset of conformationally restricted, engineered cysteines that exhibit reactivity that is dependent on the gating state of the channel. 3. To identify sites within the CFTR anion conduction pathway where the rate of covalent labeling is altered by the presence of a pore-blocking, small molecule. 4. To refine an atomic-scale model for CFTR based on the prokaryotic homologues, MsbA and Sav1866 and a two-dimensional EM map of CFTR, utilizing an iterative process of molecular modeling, quantitative model predictions and experimental testing;and to use this model to define the physical basis for anion conduction.